Research Abstracts from CPEA Investigators

Overall description: Autism is a neuro-developmental disorder with several known environmental and genetic risk factors. Converging evidence from anatomical studies, teratologic studies, and studies of comorbid conditions suggests that one cause of autism is early maldevelopment of the brain stem. Evidence for several new candidates genes for ASD susceptibility has been published in the last year, and all are genes involved in the embryonic development of the brain stem. In Project I. Animal Models of Autism and Mechanisms of Injury, we shall study the neuroanatomy of animal models and quantify gene expression in embryos exposed to teratogens, using real-time PCR and in situ hybridization. We shall extend out studies of the mechanisms of action of teratogens associated with autism to investigate the role of histone deacetylation. We shall compare transcription efficacy of polymorphisms of genetic susceptibility factors in TET-responsive cell lines. We shall look for gene-environment interactions in genetically-engineered mice exposed to teratogens in utero. The goal is to understand how teratologic agents and genetic anomalies produce similar effects on the hindbrain. Project II. Behaviors Discriminating Autism in Humans and Animals, is an investigation of behavioral tasks with promise to discriminate autism from other developmental disabilities. The goal is to develop animal tasks that can serve as markers of the developmental abnormalities that cause autism so that animal models like those developed in Project I. can be tested for parallelism to the human disorder. Inhibition of return and eyeblink conditioning are the tasks under study in this proposal. Project IV. is Gene-environment interactions in three populations exposed to teratogens associated with autism. We shall examine six suspected genetic susceptibility factors in people exposed in utero to three teratogens known to be associated with autism: thalidomide, valproic acid, and misoprostol. We shall determine whether the members of these population who meet diagnostic criteria for ASDs are ones with genetic risk factors. This Project is closely related to the gene-environment studies proposed in animals.

Project I: Animal Models of Autism and Mechanisms of Injury
Investigators: Lorraine Gudas, Ph.D., and Patrician Rodier, Ph.D.

Discovery of the embryonic stage when injury can cause autism has led to the insight that the disorder can be initiated by changes in the developing brain stem. Our studies in the first cycle support a unifying hypothesis regarding the multiple etiologies of early developmental genes are the cause of some familial cases and the teratogens that cause the disorder act by interfering with function of the same genes. A recent discovery of one of the mechanisms if valproic acid's teratogenicity opens a new avenue by which to study this drugs' effects on the developing brain. We propose to quantify the expression of Hoxa1, Hoxb 1, Gbx2 and Wnt2 after exposure of 12 day rat embryos to trichostatin A, a direct, specific inhibitor of histone deacetylase. We shall compare the morphology of adult rats exposed in utero to trichostatin A or saline. The regions to be studied sterologically are the cranial nerve motor nuclei, the cerebellum, the inferior olive, and the deep nuclei of the cerebellum. We shall evaluate by in situ hybridization the expression of RNA markers of rhombomeres in mouse embryos exposed to trichostatin A. The effects of teratogenic and non-teratogenic analogs of valproic acid will be compared to those of teratogenic and non-tertogenic enantomers of 2-ethylhexanoic acid for histone decetylase inhibition. By real-time PCR in rats and in situ hybridization in mice, we shall describe the expression of target genes of Hoxal after embryonic exposure to VPA or 2-thylhexanoic acid. The target genes to be studies are: SOD1, Gbx2, Wnt2, and EVX2. Expression levels of each of these are altered at least four-fold by overexpression of Hoxal. We shall use human mutations of early developmental genes identified in Project III to create mouse models of genetically-induced autism, and examine the morphology of the brain stem in the transgenic mutants. Subjecting heterozygotes of the gentic model to teratogens will tell us whether injury can interact with genetic abnormality to alter the brain stem. In Project IV, human populations exposed to teratogens will be tested for genotypes of suspected susceptibility genes for ASD. The animal models developed in Project I will be evaluated behaviorally in Project II. The studies of Project I will help us predict which teratogens are likely causes of autism, and further our understanding of the developmental origin of the brain abnormalities associated with ASD.

Project II: Behavioral Alterations in Autism and Rodent Models
Investigators: Susan Bryson, Ph.D., Mark Stanton, PH.D., and Jane Herbert, Ph.D.

This project will build on the success from our strategy, during the first project period, of behaviorally evaluating individuals with autism and rodent models that have been, or will be, developed and characterized neurobiologically in Project I. We emphasize tasks and experimental paradigms that discriminate autism other disorders and that have the potential to more directly address behavioral, developmental and neurological parallels between autism and our animal models. Specific Aim I will extend current studies of eyeblink conditioning in individuals with autism and in rodent models. Studies in humans will examine "higher order" variants of the procedure such as long-delay and trace conditioning, changes in conditional response (CR) timing that occur with alterations in CS-US delay intervals, and discrimination learning and reversal. Studies in rodents will examine these same eyeblink conditioning phenomena in developing rats and mice that represent various environmental and genetic models of autism form the previous and current project periods. Specific Aim 2 will pursue evidence for altered spatial inhibition in autism. Adults with autism and matched comparison groups will be tested on tasks designed to assess location-based vs. object-based inhibition (e.g. standard measures of inhibition-of-return and response inhibition) in order to better understand the specific processes underlying alterations in autism of performance on these tasks. Specific Aim 3 will further examine aspects of alterations of spatial memory and inhibition in rodent models of autism. This work will extend previous findings of alterations in some of these domains in rats gestationally exposed to valporic acid (VPA) to other rodent models. Taken together, the studies in Project II will (a) provide important, new information concerning behavioral alterations in autism; (b) determine in great detail whether these same alterations are observed in various rodent models of the etiology of this disorder; and (c) begin to reveal , with Project I, aspects of the developmental progression and underlying neurobiological mechanisms of autism.

Project IV : Gene-Environment Interactions in Autism
Principal Investigator: Patricia Rodier, Ph.D.

Three well-established environmental risk factors for autism spectrum disorders are thalidomide, valporic acid, and misoprostol. The investigators on this project have identified populations exposed to these drugs in utero. Some members of these populations have an ASD diagnosis and some do not. Most have never been tested. The hypothesis of this project is that genetic susceptibility to autism interacts with these exposures to produce the symptoms of autism in some of the people exposed, while those without genetic risk factors show fewer symptoms. To test this hypothesis we shall collect DNA sample from people with a history of exposure and genotype them for alleles of six genes thought to play a role in autism susceptibility. These are HOXA1,HOXB1, HOXD, WNT2, and GBX2. The same individuals will be screened for symptoms of ASDs, and those failing the screen will be tested by the ADI/ADOS. We shall then determine whether the alleles of each gene occur in affected and unaffected individuals at rates expected by chance, or whether the rates differ from chance. Participants who meet criteria for an ASD diagnosis will be further characterized as to language, cognition, neurobiological dysfunctions, physical malformations, and adaptive function, so that the phenotypes associated with the three exposures can be compared with each other, and with ASDs of unknown cause. Investigation of gene-environment interactions has led to breakthroughs in other complex genetic disorders, such as neural tube defects. This will be the first attempt to apply this approach to ASDs.